Effect of oxygen on the non-photochemical quenching of vascular plants and potential oxygen deficiency in the stroma of PsbS-knock-out rice.

The excessive and harmful light energy absorbed by the photosystem (PS) II of higher plants is dissipated as heat through a protective mechanism termed non-photochemical quenching (NPQ) of chlorophyll fluorescence. PsbS-knock-out (KO) mutants lack the trans-thylakoid proton gradient (ΔpH)-dependent part of NPQ. To elucidate the molecular mechanism of NPQ, we investigated its dependency on oxygen. The development of NPQ in wild-type (WT) rice under low-oxygen (LO) conditions was reduced to more than 50% of its original value. However, under high-oxygen (HO) conditions, the NPQ of both WT and PsbS-KO mutants recovered. Moreover, WT and PsbS-KO mutant leaves infiltrated with the ΔpH dissipating uncoupler nigericin showed increased NPQ values under HO conditions. The experiments using intact chloroplasts and protoplasts of Arabidopsis thaliana supported that the LO effects observed in rice leaves were not due to carbon dioxide deficiency. There was a noticeable 90% reduction in the half-time of P700 oxidation rate in LO-treated leaves compared with that of WT control leaves, but the HO treatment did not significantly change the half-time of P700 oxidation rate. Overall, the results obtained here indicate that the stroma of the PsbS-KO plants could be potentially under O2 deficiency. Because the functions of PsbS in rice leaves are likely to be similar to those in other higher plants, our findings offer novel insights into the role of oxygen in the development of NPQ.

Rice mutants deficient in ω-3 fatty acid desaturase (FAD8) fail to acclimate to cold temperatures.

To investigate the role of ω-3 fatty acid (FA) desaturase (FAD8) during cold acclimation in higher plants, we characterized three independent T-DNA insertional knock-out mutants of OsFAD8 from rice (Oryza sativa L.). At room temperature (28 °C), osfad8 plants exhibited significant alterations in fatty acid (FA) unsaturation for all four investigated plastidic lipid classes. During a 5-d acclimation period at 4 °C, further changes in FA unsaturation in both wild-type (WT) and mutant plants varied according to the type of lipid. We also monitored the fluidity of the thylakoid membrane using a threshold temperature to represent the change in fluorescence. The values were altered significantly by both FAD8 mutation and cold acclimation, suggesting that factors other than FAD8 are involved in C18 FA unsaturation and fluctuations in membrane fluidity. Similarly, significant changes were noted for both the mutant and WT samples in terms of their FA compositions as well as activities related to photosystem (PS) I, PSII, and photoprotection. This included the development of non-photochemical quenching and increased zeaxanthin accumulation. Despite the relatively small changes in FA composition during cold acclimation, cold-inducible FAD8 knock-out mutants displayed strong differences in photoprotective activities and a further drop in membrane fluidity. The mutants were more sensitive than WT to short-term low-temperature stress that resulted in increased production of reactive oxygen species after 5 d of chilling. Taken together, our findings suggest that FA unsaturation by OsFAD8 is crucial for the acclimation of higher plants to low-temperature stress.

Production of superoxide from Photosystem II in a rice (Oryza sativa L.) mutant lacking PsbS.

BACKGROUND: PsbS is a 22-kDa Photosystem (PS) II protein involved in non-photochemical quenching (NPQ) of chlorophyll fluorescence. Rice (Oryza sativa L.) has two PsbS genes, PsbS1 and PsbS2. However, only inactivation of PsbS1, through a knockout (PsbS1-KO) or in RNAi transgenic plants, results in plants deficient in qE, the energy-dependent component of NPQ. RESULTS: In studies presented here, under fluctuating high light, growth of young seedlings lacking PsbS is retarded, and PSII in detached leaves of the mutants is more sensitive to photoinhibitory illumination compared with the wild type. Using both histochemical and fluorescent probes, we determined the levels of reactive oxygen species, including singlet oxygen, superoxide, and hydrogen peroxide, in leaves and thylakoids. The PsbS-deficient plants generated more superoxide and hydrogen peroxide in their chloroplasts. PSII complexes isolated from them produced more superoxide compared with the wild type, and PSII-driven superoxide production was higher in the mutants. However, we could not observe such differences either in isolated PSI complexes or through PSI-driven electron transport. Time-course experiments using isolated thylakoids showed that superoxide production was the initial event, and that production of hydrogen peroxide proceeded from that. CONCLUSION: These results indicate that at least some of the photoprotection provided by PsbS and qE is mediated by preventing production of superoxide released from PSII under conditions of excess excitation energy.

Acceleration of cyclic electron flow in rice plants (Oryza sativa L.) deficient in the PsbS protein of Photosystem II.

When compared with Photosystem I (PSI) in wild-type (WT) rice plants, PSI in PsbS-knockout (KO) plants that lack the energy-dependent component of nonphotochemical quenching (NPQ) was less sensitive to photoinhibition. Therefore, we investigated the relationship between NPQ and cyclic electron flow (CEF) around PSI as a photoprotective mechanism. Activities of two CEF routes (PGR5-dependent or NDH-dependent) were compared between those genotypes by using both dark-adapted plants and pre-illuminated plants, i.e., those in which the Calvin-Benson cycle is de-activated and activated, respectively. In dark-adapted leaves activity of the PGR5-dependent route was determined as the rate of P700 photooxidation. Activity was higher in the mutants than in the WT. However, no difference was noted when plants of either genotype were pre-illuminated. When the electron transport pathway was switched to the cyclic mode by infiltrating leaf segments with 150 mM sorbitol, 40 µM DCMU, and 2 mM hydroxylamine, the rate of P700 oxidation was faster in the mutant. That difference disappeared when leaves were infiltrated with antimycin A to inhibit the PGR5-dependent route. Chlorophyll fluorescence (Fo) was also evaluated. To achieve an Fo level comparable to that of the WT, activation of the NDH-dependent route in the mutant required pre-illumination at a certain dose. Therefore, we propose that, as an alternate pathway for the photoprotection of photosystems in the absence of energy-dependent quenching, this PGR5-dependent route is more highly activated in the PsbS-KO mutants than in the WT. Moreover, that stronger activity is probably responsible for slower activation of the NDH-dependent route in the mutant.

Loss-of-function of OsSTN8 suppresses the photosystem II core protein phosphorylation and interferes with the photosystem II repair mechanism in rice (Oryza sativa).

STN8 kinase is involved in photosystem II (PSII) core protein phosphorylation (PCPP). To examine the role of PCPP in PSII repair during high light (HL) illumination, we characterized a T-DNA insertional knockout mutant of the rice (Oryza sativa) STN8 gene. In this osstn8 mutant, PCPP was significantly suppressed, and the grana were thin and elongated. Upon HL illumination, PSII was strongly inactivated in the mutants, but the D1 protein was degraded more slowly than in wild-type, and mobilization of the PSII supercomplexes from the grana to the stromal lamellae for repair was also suppressed. In addition, higher accumulation of reactive oxygen species and preferential oxidation of PSII reaction center core proteins in thylakoid membranes were observed in the mutants during HL illumination. Taken together, our current data show that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants and to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8-mediated PCPP in PSII repair.

Correlations between the temperature dependence of chlorophyll fluorescence and the fluidity of thylakoid membranes.

To monitor changes in membrane fluidity in Arabidopsis leaves and thylakoid membranes, we investigated the temperature dependence of a chlorophyll fluorescence parameter, minimum fluorescence (Fo), and calculated the threshold temperature [T(Fo)] at which the rise of the fluorescence level Fo was considered to be started. For the modification of membrane fluidity we took three different approaches: (1) an examination of wild-type leaves initially cultured at room temperature (22°C), then exposed to either a lower (4°C) or higher (35°C) temperature for 5 days; (2) measurements of the shift in T(Fo) by two mutants deficient in fatty acid desaturase genes - fad7 and fad7fad8 and (3) an evaluation of the performance of wild-type plants when leaves were infiltrated with chemicals that modify fluidity. When wild-type plants were grown at 22°C, the T(Fo) was 48.3 ± 0.3°C. Plants that were then transferred to a chamber set at 4 or 35°C showed a shift in their T(Fo) to 42.7 ± 0.9°C or 48.9 ± 0.1°C, respectively. Under low-temperature acclimation, the decline in this putative transition temperature was significantly less in fad7 and fad7fad8 mutants compared with the wild-type. In both leaf and thylakoid samples, values for T(Fo) were reduced in samples treated with benzyl alcohol, a membrane fluidizer, whereas T(Fo) rose in samples treated with dimethylsulfoxide, a membrane rigidifier. These results indicate that the heat-induced rise of chlorophyll fluorescence is strongly correlated with the fluidity of thylakoid membranes.

Acute exposure to UV-B sensitizes cucumber, tomato, and Arabidopsis plants to photooxidative stress by inhibiting thermal energy dissipation and antioxidant defense.

To characterize a change in NPQ upon exposure to ultraviolet-B (UV-B), the xanthophyll cycle-dependent and -independent NPQs were compared in Cucumis sativus, Lycopersicum esculentum, and Arabidopsis thaliana leaves. The xanthophyll cycle-dependent NPQ was dramatically but reversibly suppressed by UV-B radiation. This suppression was correlated more strongly with a marked decrease in photosynthetic electron transport rather than changes in xanthophyll cycle enzymes such as violaxanthin de-epoxidase and zeaxanthin epoxidase. Accordingly, the UV-B-induced suppression of NPQ cannot be attributed to changes in expressions of VDE and ZEP. However, suppression of the xanthophyll cycle-dependent NPQ could only account for the 77 K fluorescence emission spectra of thylakoid membranes and the increased level of (1)O(2) production, but not for the decreased levels of •O(2)(-) production and H(2)O(2) scavenging. These results suggest that a gradual reduction of H(2)O(2) scavenging activity as well as a transient and reversible suppression of thermal energy dissipation may contribute differentially to increased photooxidative damages in cucumber, tomato, and Arabidopsis plants after acute exposure to UV-B radiation.

PsbS-specific zeaxanthin-independent changes in fluorescence emission spectrum as a signature of energy-dependent non-photochemical quenching in higher plants.

The PsbS protein of photosystem II is necessary for the development of energy-dependent quenching of chlorophyll (Chl) fluorescence (qE), and PsbS-deficient Arabidopsis plant leaves failed to show qE-specific changes in the steady-state 77 K fluorescence emission spectra observed in wild-type leaves. The difference spectrum between the quenched and un-quenched states showed a negative peak at 682 nm. Although the level of qE development in the zeaxanthin-less npq1-2 mutant plants, which lacked violaxanthin de-epoxidase enzyme, was only half that of wild type, there were no noticeable changes in this qE-dependent difference spectrum. This zeaxanthin-independent DeltaF682 signal was not dependent on state transition, and the signal was not due to photobleaching of pigments either. These results suggest that DeltaF682 signal is formed due to PsbS-specific conformational changes in the quenching site of qE and is a new signature of qE generation in higher plants.